EP0839536A1 - Methoden und Zusammensetzungen zur Förderung der Immunverstärkung - Google Patents

Methoden und Zusammensetzungen zur Förderung der Immunverstärkung Download PDF

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EP0839536A1
EP0839536A1 EP98100138A EP98100138A EP0839536A1 EP 0839536 A1 EP0839536 A1 EP 0839536A1 EP 98100138 A EP98100138 A EP 98100138A EP 98100138 A EP98100138 A EP 98100138A EP 0839536 A1 EP0839536 A1 EP 0839536A1
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cells
protein
immunopotentiating
composition
cell
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Jeffrey A. Dr. Bluestone
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Arch Development Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to immunology, and, more specifically, to the preparation and use of immunopotentiating agents which are capable of eliciting, enhancing and/or otherwise modifying immune responses. These agents, through their ability to elicit or enhance cellular or humoral responses, have potential utility in a variety of disease conditions wherein immunotherapy might be expected to provide a benefit.
  • the body's immune system serves as a defense against a variety of conditions, including, e.g., injury, infection and neoplasia, and is mediated by two separate but interrelated systems, the cellular and humoral immune systems.
  • the humoral system is mediated by soluble products, termed antibodies or immunoglobulins, which have the ability to combine with and neutralize products recognized by the system as being foreign to the body.
  • the cellular immune system involves the mobilization of certain cells, termed T-cells, that serve a variety of therapeutic roles.
  • T cells thymus derived cells
  • B cells bone marrow derived cells
  • Mature T cells emerge from the thymus and circulate between the tissues, lymphatics, and the bloodstream. T cells exhibit immunological specificity and are directly involved in cell-mediated immune responses (such as graft rejection). T cells act against or in response to a variety of foreign structures (antigens). In many instances these foreign antigens are expressed on host cells as a result of infection. However, foreign antigens can also come from the host having been altered by neoplasia or infection. Although T cells do not themselves secrete antibodies, they are usually required for antibody secretion by the second class of lymphocytes, B cells.
  • T cells There are various subsets of T cells, which are generally defined by antigenic determinants found on their cell surfaces, as well as functional activity and foreign antigen recognition. Some subsets of T cells, such as CD8 + cells, are killer/suppressor cells that play a regulating function in the immune system, while others, such as CD4 + cells, serve to promote inflammatory and humoral responses.
  • CD refers to cell differentiation cluster; the accompanying numbers are provided in accordance with terminology set forth by the International Workshops on Leukocyte Differentiation (5). A general reference for all aspects of the immune system may be found in (1)).
  • Human peripheral T lymphocytes can be stimulated to undergo mitosis by a variety of agents including foreign antigens, monoclonal antibodies and lectins such as phytohemaggluttinin and concanavalin A. Although activation presumably occurs by binding of the mitogens to specific sites on cell membranes, the nature of these receptors, and their mechanism of activation, is not completely elucidated. Induction of proliferation is only one indication of T cell activation. Other indications of activation, defined as alterations in the basal or resting state of the cell, include increased lymphokine production and cytotoxic cell activity.
  • T cell activation is an unexpectedly complex phenomenon that depends on the participation of a variety of cell surface molecules expressed on the responding T cell population (2,3).
  • the antigen-specific T cell receptor (TcR) is composed of a disulfide-linked heterodimer, containing two clonally distributed, integral membrane glycoprotein chains, ⁇ and ⁇ , or ⁇ and ⁇ , non-covalently associated with a complex of low molecular weight invariant proteins, commonly designated as CD3 (the older terminology is T3) (2,4).
  • the TcR ⁇ and ⁇ chains determine antigen specificities (6).
  • the CD3 structures are thought to represent accessory molecules that may be the transducing elements of activation signals initiated upon binding of the TcR ⁇ to its ligand.
  • Polymorphic TcR variable regions define subsets of T cells, with distinct specificities.
  • the TcR complex interacts with small peptidic antigen presented in the context of major histocompatability complex (MHC) proteins.
  • MHC proteins represent another highly polymorphic set of molecules randomly dispersed throughout the species. Thus, activation usually requires the tripartite interaction of the TcR and foreign peptidic antigen bound to the major MHC proteins.
  • Monoclonal antibodies were developed by Kohler and Milstein in 1975.
  • the methods generally used to produce mAb consist of fusing (hybridizing) two types of somatic cells: (1) a neoplastic myeloma cell line; and (2) a normal B lymphocyte obtained from an immunized animal.
  • the result is called a hybridoma which is characterized by immortal growth and the ability to secrete antibodies specific for the immunization antigen.
  • One of the clinically successful uses of monoclonal antibodies is to suppress the immune system, thus enhancing the efficacy of organ or tissue transplantation.
  • U.S. Patent 4,658,019 describes a novel hybridoma (designated OKT3) which is capable of producing a monoclonal antibody against an antigen found on essentially all normal human peripheral T cells. This antibody is said to be monospecific for a single determinant on these T cells, and does not react with other normal peripheral blood lymphoid cells.
  • the OKT3 mAb described in this patent is currently employed to prevent renal transplant rejection (10).
  • OKT3 has not been previously used to stimulate the immune system in vivo .
  • T cell surface molecules have been identified that can activate T cell function, but are not necessarily part of the T cell surface receptor complex.
  • Monoclonal antibodies against Thy-1, TAP, Ly-6, CD2, or CD28 molecules can activate T cells in the absence of foreign antigen in vitro (12, 13,14,15,16).
  • certain bacterial proteins although differing in structure from mAbs, also have been shown to bind to subsets of T cells and activate them in vitro (17). Although some of these agents, in vitro effects have previously been demonstrated, in vitro activity is often not a reliable predictor of in vitro effects.
  • malignant tumor growth is believed to be the inability of the immune system to respond appropriately to tumor antigen.
  • malignant progressor tumors are only weakly immunogenic and can evade host recognition and rejection. Both specific and non-specific effector pathways have been implicated in tumor immunity.
  • Treatment by immunotherapy is aimed at remedying defects in the immune weaponry.
  • the aim of immunotherapy has been the enhancement of one or both of these pathways.
  • One potential approach to therapy is to activate host antitumor cellular effector mechanisms.
  • non-specific adjuvants such as BCG or pertussis have been used to augment immune responses.
  • these adjuvants amplify immune responses by providing non-specific stimuli that enhance overall immunity.
  • these adjuvants do not selectively act on T cells, or subsets of T cells, and have not been shown capable of overcoming immunodeficiency states.
  • current modes of immunotherapy which induce non-specific effector cells are not effective enough in potentiating anti-tumor responses (18).
  • immunotherapy regimens which utilize the ability of the immune system to recognize tumor antigens in a specific manner, for instance utilizing specific tumor-infiltrating lymphocytes, for immunotherapy, have been suggested to result in superior anti-tumor immunity (19).
  • HIV Human immunodeficiency virus
  • HIV Human immunodeficiency virus
  • humoral and cell mediated effector mechanisms Although current efforts in treatment and vaccine design have fallen short of success either due to the immunodeficiency associated with the viral infection, or to the low immunogenicity of the vaccine (20).
  • the development of a safe and effective vaccine against infection with human immunodeficiency virus (HIV) is complicated by a lack of understanding of protective immunity to HIV and disease development, and the absence of an adequate and convenient animal model for studying HIV infection.
  • HIV can be transmitted as either a cell-free or cell-associated virus
  • a protective immune response against HIV infection will likely require both humoral and cell-mediated immunity, including neutralizing antibody against HIV, antibodies involved in antibody-dependent cellular cytotoxicity and cytotoxic lymphocytes. All of these activities involve virus-specific T cells. T cell activation requires potent in vitro immune responses to foreign antigens such as viruses.
  • CD4 is a membrane protein that acts as a binding site and entry port into CD4 + lymphocytes for the human immunodeficiency virus-type 1 (HIV-1) (21).
  • MHC major histocompatibility antigens
  • Any vaccine approach which utilizes HIV peptides or inactivated virus antigen must depend on the ability of antigenic peptides to bind the appropriate MHC antigens necessary to initiate an immune response. Given the tremendous polymorphism of the MHC antigens expressed in the population, and the variation of the HIV virus, developing a successful HIV vaccine for general use is difficult and has not yet been successful.
  • peptides, proteins or other potential or desired immunogens in vaccines can be limited by several critical factors.
  • low immunogenicity of the peptide or other structure which one desires to employ can be a difficult problem to overcome, particularly with smaller peptides and those peptides which do not contain appropriately strong B- and/or T-cell potentiating sequences.
  • Such peptides are typically only weakly immunogenic at best.
  • the peptides chosen must be capable of inducing an immune response in a majority of the population.
  • compositions and/or methodology for eliciting or enhancing cellular or humoral responses in mammals are needed both to provide animal models for investigation of therapeutic regimes, to provide novel means of preparing improved immune system-directed products such as improved immunotherapeutic antibodies, and to advance treatment and possible immunization, e.g., for conditions such as HIV, cancer and infections.
  • the present invention is concerned with a broad array of embodiments, generally involving methods and compositions for potentiating one or more aspects of the immune response of a human or other animal having an immune system, as well as to products which may be derived out of the use of these methods and compositions.
  • the invention concerns essentially four categories of what may be referred to broadly as immunopotentiating or immunoactivating compositions: 1) individual immunopotentiating agents which are used to potentiate one or more aspects of the immune system; 2) immunopotentiating "adjuvant" compositions wherein immunopotentating agents are employed essentially as "adjuvants" to improve the body's immune responsiveness to other compounds which are co-administered with, or included in with, compositions containing the immunopotentiating agent; 3) immunopotentiating conjugates wherein the immunopotentiating agent is actually chemically coupled to the compound against which an immune response is desired; and 4) products derived from the administration of one or more of the foregoing, including, e.g., antibodies, antibody-producing cells, T-cells, potentiated bone marrow progenitor cells, and the like.
  • activation is generally defined to refer to any change induced in the basal or resting state of T or B cells. This includes, but is not limited to, increased cell proliferation and DNA synthesis, lymphokine and cytotoxic cell production, a rapid rise in intracellular calcium, release of water soluble inositol phosphates, increased IL-2 receptor expression, enhanced proliferative response to IL-2, and enhanced responses to foreign antigens or MHC (23).
  • immunopotentiating is classically defined as the ability to produce an effect on the immune system which enhances the system's ability to respond to foreign antigens. Thus, immunopotentiation may affect the cellular response, humoral response, or both.
  • Exemplary indices of immunopotentiation include, but are not limited to, cell proliferation, increased DNA synthesis, increased production of lymphokines, increased production of cytotoxic cells, calcium efflux, or any other change that raises the cell above the basal or resting state.
  • the immunopotentiation achieved by the methods and agents of this invention may affect all T cells, certain subsets of T cells, or B cells, depending on the nature of the agent(s) and their dose levels.
  • One of the objects of this invention is to provide means for fine tuning immunopotentiation, allowing one to target T and/or B cell response depending on the nature of the clinical condition to be treated.
  • the present invention concerns the preparation and use of immunopotentiation agents, whether used alone as a direct immunopotentiation agent, or combined with other compounds, either covalently or simply admixed in the same composition.
  • immunpotentiation agent is intended to include immunopotentiating antibodies, as well as certain bacterial proteins which have been determined to have profound immunopotentation actions.
  • useful immunopotentiation agents will generally involve antibodies against a cell surface epitope of T-cells wherein binding of the antibody to the surface epitope of the T-cell will result in immunopotentiation.
  • An exemplary antibody is anti-CD3 (e.g., OKT3), previously known only to be immunosuppressive and not previously known to be immunopotentiating.
  • anti-CD3 e.g., OKT3
  • the present inventor has surprisingly discovered that, in fact, when anti-CD3 is administered at relatively low doses (e.g., on the order of 100 to 200 ⁇ g/kgm body weight), rather than being immunosuppressive it exhibits a very profound immunopotentiation effect. The reason for this appears to include but may not be limited to the induction of lymphokines, the proliferation of T cells, or even the progression of T cells from a naive to memory state.
  • anti-CD3 is a useful immunopotentiation agent
  • numerous other immunopotentiation antibodies are contemplated to be within the scope of the present invention.
  • Such antibodies are defined generally as antibodies which recognize and activate a T cell activation molecule or epitope on the cell surface of T cells.
  • monoclonal antibodies which are particularly useful in the practice of the present invention will comprise those directed against the T cell variable or constant epitopes on the cell surface of T cells.
  • the T cell activation molecules which are expressed on the cell surface may be either those associated with the T cell receptor complex or those with the antigens distinct from, that is not physically associated with, TcR on the cell surface.
  • Specific embodiments of T cell activation molecules comprise either the variable or the constant region epitopes as expressed on the antigen specific T cell receptor polymorphic chains, e.g., ⁇ , ⁇ , ⁇ , and ⁇ chains.
  • Embodiments of the non-polymorphic TcR associated CD3 chains against which monoclonal antibodies may be directed for use as immunopotentiating agents are the ⁇ , ⁇ , ⁇ or ⁇ chains. Monoclonal antibodies have been developed against some of these chains, as exemplified by OKT3, SP34, UCHTI or 64.1 (68-70).
  • T cell surface antigens which are distinct from, and not physically associated with, TcR, are CD2, CD28, Thy-1, and the activation epitopes expressed on members of the Ly-6 protein family.
  • the immunopotentiation agents of the present invention will also include certain potentiating bacterial proteins such as bacterial enterotoxins, exemplified by staphylococcal enterotoxin B (SEB).
  • SEB is now known to activate T-cells and provide surprisingly profound and subset-specific potentiation.
  • the mechanism of how enterotoxins function to stimulate the immune system is not entirely clear, but could involve lymphokine production or T cell proliferation.
  • SEB comprises a preferred enterotoxin for immunopotentation purposes
  • the invention contemplates that other similar enterotoxins, such as staphylococcal enterotoxins A, C 1 , C 2 , D, E, toxic shock syndrome toxin (TSST), exfoliating toxin (ExFT) and likely even mycoplasma arthriditis substance, will find similar utility.
  • enterotoxins A staphylococcal enterotoxins A, C 1 , C 2 , D, E
  • TSST toxic shock syndrome toxin
  • ExFT exfoliating toxin
  • mycoplasma arthriditis substance likely even mycoplasma arthriditis substance
  • a pharmacologically suitable vehicle such as combining with an appropriate diluent or buffer
  • an appropriate diluent or buffer in an amount and concentration that is appropriate to effectuate potentiation of the immune system when administered.
  • parenteral compositions having from about 0.1 mg/ml to about 1 mg/ml, in order to allow the parenteral administration of appropriate amounts of the antibody.
  • the present invention contemplates the use of these immunopotentiating agents in immunogen containing compositions such as vaccines, where the agents serve as "adjuvants" to improve the immunogenicity of other components of the composition.
  • the agents serve as "adjuvants" to improve the immunogenicity of other components of the composition.
  • the immunopotentiating protein is admixed or otherwise coadministered with the molecule against which an immune response is desired, with the immunopotentiating agent being present in amounts effective to promote potentiation upon administration to the particular subject.
  • an immunopotentiating heteroconjugate is defined as an immunopotentiating agent conjugated to a second protein or other molecule against which an immune response is desired, the conjugation being in the nature of a chemical or molecular crosslink.
  • Exemplary embodiments of the second molecule include proteins which comprise amino acid sequences or other potential determinants (e.g., a non-amino acid determinant such as a sialo group) against which a cellular or humoral immune response is desired.
  • a non-amino acid determinant such as a sialo group
  • the present invention also contemplates the use of non-protein molecules, such as glycolipids, carbohydrates or even lectins, as the second molecule against which an immune reaction is sought. All that is required for use in the context of heterconjugates is that one be capable of conjugating the molecule to the immunopotentiation agent.
  • the immunopotentiating protein of the heteroconjugate may also be formed by linking two monoclonal antibodies directed against two distinct but specific T cell epitopes.
  • a specific embodiment of this type of immunopotentiating protein is anti-CD3 coupled with anti-CD4 to form a CD4 subset-specific immunopotentiating protein. It has been demonstrated that administering of such a bispecific antibody construct in vivo activates T cells to a surprising degree (see, e.g., U.S. Patent 4,676,980 to Segal, and Figure 14 herein). It is proposed by the present inventor that one may employ such a bispecific antibody construct as the immunopotentiating ligand of heteroconjugates formed with a second protein against which an immune response is desired.
  • heteroconjugate disclosed in this invention is believed to aid in directing the immune attack to specific epitopes, via the attached compound or protein.
  • a particular utility of these embodiments will be found in tee treatment of diseases such as cancer, where only weakly immunogenic tumor-specific or tumor-associated proteins are found to appear on the cell surface of many tumors, or are found to characterize the tumor.
  • the epitopes used as second proteins may either be derived from the cell surface or extracted from cells which are infected or diseased.
  • amino acid sequences can be made synthetically by standard chemical synthetic or molecular biology methods.
  • amino acid sequences are known for various peptides isolated from the HIV virus gp120 cell membrane which can be employed as embodiments of the second compound, whether it be conjugated to the immunopotentiating protein or simply admixed in an appropriate pharmaceutical vehicle.
  • Preferred embodiments of peptides which may be used as second proteins in a heteroconjugate used to augment immunity against HIV comprise peptides 18, T1, T2, or peptides derived from the CD4 binding site.
  • epitopes specific for amino acid sequences homologous with those expressed on the surface of human hepatitis virus, or extracted from cells infected with the virus may be used as the second protein.
  • Viral specific epitopes may also comprise amino sequences homologous with those expressed on the surface of infected cells.
  • Immunogenic compositions may be prepared for administration to subjects by including any of the foregoing immunopotentiating compositions, and formulated to include an effective amount of 1) an immunopotentiating protein (i.e., where one desires simply generalized immunopotentiation rather than potentiation that is directed against a second compound), 2) an immunopotentiation protein in combination with a second compound, or 3) an immunopotentiation protein conjugated to such a second compound.
  • the materials that are formulated are preferably rendered pharmacologically acceptable by manufacturing in accordance with good manufacturing practices, extensively dialyzed to remove undesirable small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.
  • compositions are intended for human administration, one will typically desire to include an amount of immunopotentiation agent that will result in T cell activation in the absence of concommitant immunosuppression.
  • the determination of exact amounts will depend on the particular circumstances, such as the particular condition to be treated, the physical condition of the patient, the type of immunogenic composition that is to be administered, and the like.
  • suitable formulations should typically include from about 10 ug to about 1000 ug bolus/patient every 14 days or so, and more preferably 100 ug to about 400 ug of the antibody per patient.
  • enterotoxins that one should typically desire to employ from about 100 ug to about 10 mg of the enterotoxin per. dosage, and more preferably about 1 to about 5 mg/dose.
  • the immunogens of the invention may be formulated in oils such as sesame or peanut oil, aqueous propylene glycol, in liposomes or in sterile aqueous solutions. Such solutions are typically suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Additionally, stabilizers in the form of, for example, sorbitol or stabilized gelatin may be included. These particular aqueous solutions may be particularly well suited for intramuscular and subcutaneous injections, as may be preferred for vaccination using antigenic preparations.
  • the proteins may be formulated into the composition as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isoprophylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • quantity to be administered depends on the subject to be treated, including, e.g. , the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimens for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • Various methods of achieving additional adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70 ° to 101 ° C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C.
  • agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70 ° to 101 ° C for 30 second to 2 minute periods respectively. Aggregation by reactivating with peps
  • parvum or endotoxins or lipopolysacchraide components of gram-negative bacterial, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • the invention is by no means limited to human application, and is intended to apply to any mammal having an immune system, including, e.g., rodents such as mice, hamsters and rats, primates, rabbits, even farm animals such as cows, pigs, etc.
  • rodents such as mice, hamsters and rats, primates, rabbits, even farm animals such as cows, pigs, etc.
  • the invention contemplates, e.g., that certain of the foregoing embodiments will have general applicability wherever one desires to obtain an enhanced immune response against a desired molecule, such as in the initial immunization of animals for hybridoma or even polyclonal antibody development.
  • the nature of the second molecule is not crucial to the successful practice of the invention, it is recognized that the invention will find its greatest utility where the second molecule is a peptide, in that peptides are often notoriously difficult to obtain an immune response against. Thus, it is believed that particular benefits will be realized through the use of peptides having from about 8 to about 100 amino acids in length, and even more preferably, about 8 to about 50 amino acids in length.
  • heteroconjugates In the context of heteroconjugates, it is contemplated that numerous methods for conjugation may be applied, including but not limited to: 1) the formation of biotin-avidin bridges; 2) the use of cross linkers such as SPDP to link the functional units; 3) cross-linking of maleimide and SH groups; as well as numerous other possibilities. In general, all that is required is that the cross-linking maintain the integrity of the peptidic antigen and leave unaltered the activating property of the immunopotentiating reagent.
  • the present invention contemplates that various useful biological products may be derived through the application of the foregoing immunopotentiating compositions.
  • the adjuvant and heteroconjugate embodiments will provide extremely useful means for preparing antibodies, including monclonal antibodies.
  • immunopotentiating antibodies such as anti-CD3 can serve to promote the recruitment of hematopoetic progenitor cells, presumably by stimulating the release of cytokines and lymphokines from activated T-cells.
  • activated T cells themselves will find some utility, such as in anti-tumor therapy that employs tumor-infiltrating lymphocytes.
  • FIGURE 1 Forms of Immune Augmentation .
  • This figure demonstrates an overview of various embodiments of the invention, including "immunoadjuvant" embodiments wherein the immunopotentiating protein is simply admixed with a compound against which an immune response is desired (top panel), or where and actual heteroconjugate is formed between the immunopotentiating protein and the compound (middle panel).
  • top panel a compound against which an immune response is desired
  • the immunopotentiating ligand is actually a bifunctional cojugate formed between two antibodies.
  • FIGURE 2 Activation of peripheral lymph node T cells from anti-CD3-treated C3H mice as assessed by flow cytometry (FCM) .
  • FCM flow cytometry
  • Two color FCM from control animals and those treated with, 4, 40, or 400 ⁇ g of anti-CD3 are displayed as contour plots on a logarithmic scale. Intensity of green FITC fluorescence is plotted along the x-axis and red (B-phycoerythrin) fluorescence is plotted along the y-axis.
  • A Anti-CD4 staining on the x-axis and anti-IL-2 receptor (Il-2R) staining on the y-axis.
  • FIGURE 3 In vitro proliferation of lymph node T cells from anti-CD3 treated C3H mice . Eighteen hours after intravenous anti-CD3 administration, lymph nodes were removed from animals and the cells (1 x 10 5 cells for mixed lymphocyte reaction (MLR) and mixed lymphocyte tumor culture (MLTC)) were incubated in medium that contained irradiated syngeneic spleen cells (2 x 10 5 ) plus recombinant IL-2 (rIL-2), or irradiated allogeneic (C57BL/10) spleen cells, or mitomycin C-treated Pro-4L tumor cells (5 x10 3 ).
  • MLR mixed lymphocyte reaction
  • MLTC mixed lymphocyte tumor culture
  • Proliferation was measured by [ 3 H]thymidine uptake at 48 hours (rIL-2) or 72 hours (MLR and MLTC). Background uptake (generally less than 5 x 10 3 cpm) was determined from lymph node cells stimulated with syngeneic irradiated accessory cells, or from mitomycin C-treated tumor cells cultured alone, and was subtracted from values for treated cells. All assays were performed in triplicate; standard deviations were less than 5%. The results show that a) functional IL-2R is expressed on anti-CD3 treated cells; and b)anti-CD3 treated T cells responded more vigorously to allogeneic MHC and tumor antigens than did untreated cells under otherwise similar conditions.
  • FIGURE 4 Colony Stimulating Factor (CSF) in serum of mice after injection of anti-CD3 .
  • CSF Colony Stimulating Factor
  • FIGURE 5 Clinical Response to anti-CD3 (OKT3) Treatment .
  • FIGURE 6 Anti-CD3 augments immune response .
  • Anti-KLH Keyhole limpet hemocyanin
  • FIGURE 7 Alloresponse of lymphocytes from C3H mice treated with Staphylococcus enterotoxin B (SEB) or mAb 145-2C11 .
  • SEB Staphylococcus enterotoxin B
  • mAb 145-2C11 anti-CD3
  • FIGURE 7 Alloresponse of lymphocytes from C3H mice treated with Staphylococcus enterotoxin B (SEB) or mAb 145-2C11 .
  • SEB Staphylococcus enterotoxin B
  • mAb 145-2C11 anti-CD3
  • FIGURE 87 IL-2R expression on T cells from SEB-treated mice . Mice were treated with increasing doses of SEB (0, 5, 50, 250 ⁇ g). Il-2R expression after 18 hours was compared using flow cytometry and showed enhanced expression. Dose response was observed.
  • FIGURE 9 Proliferative response to SEB . Lymph node cell proliferation was assayed by 3 H uptake (CPM) in C3H mice at 18 hours after administration of SEB. rIL-2 response to SEB 3 H was enhanced compared to controls or to 145-2C11-treated mice. In addition, there was a dose response (5, 50, 250 ⁇ g of SEB).
  • CPM 3 H uptake
  • FIGURE 10 Expansion of V ⁇ 8 + cells in SEB-treated mice . Three days after treatment of mice with SEB, spleen cells were incubated with anti-V ⁇ 8 and V ⁇ 8 + cells were assayed by flow cytometry. Expansion of V ⁇ 8 + cells was observed due to SEB treatment.
  • FIGURE 11 Distribution of lymphocyte subsets after treatment with low doses of OKT3 .
  • OKT3 100 ⁇ g/patient
  • IL-2 receptor CD25
  • FIGURE 12 Increased lymphokine production after treatment of humans with OKT3 .
  • relative levels of hematopoetic progenitor cells e.g., bands, myelocytes
  • hematopoetic progenitor cells e.g., bands, myelocytes
  • FIGURE 13 Survival of grafts as a function of treatment with anti-CD3 .
  • GVHD graft versus host disease
  • FIGURE 14 Effects of treatment of mice in vivo with a combination of 1005/45-KLH and 145-2C11 .
  • This figure reflects studies conducted to demonstrate the adjuvant characteristics of anti-CD3 (mAb 145-2C11, an anti-murine CD3 mAb specific for a 25 kd protein CD3- ⁇ ) when co-administered with a KLH-linked peptide (1005/45; ref. 29), when administered intraperitoneally.
  • C57BL/10 mice were administered 100 ⁇ g of the 1005/45-KLH conjugate, intraperitoneal (IP) or subcutaneously (SC), either alone or co-administered with 4 ug of anti-CD3 (145-2C11) IP.
  • IP intraperitoneal
  • SC subcutaneously
  • FIGURE 15 In vivo treatment with mAbs .
  • the response of IL-2 receptor positive cells was compared after treatment with anti-CD3 alone versus after treatment with a heteroconjugate comprising anti-CD3 and anti-CD4.
  • Anti-CD3 alone resulted in significant increases in both CD4+ and CD8+ IL-2 receptor+ cells.
  • the heteroconjugate (linked by the SPDP method) produced an enhanced IL-2R expression on CD4+ cells, indicative of a subset-specific response.
  • FIGURE 16 In vivo immune stimulation of mice administered a heteroconjugate .
  • Panel A IgG anti-FITC antibody production in anti-CD3 treated mice immunized with FITC-BSA in complete Freunds adjuvant (CFA) or PBS, compared to ELISA measurements of sera from control mice (open bars).
  • Panel B IgG anti-FITC antibody production measured from sera of FITC-anti-CD3 treated mice (left-hatched bars) compared to FITC-normal Hamster Ig (cross-hatched bars) measured by ELISA performed on day 10 bleeds.
  • FIGURE 17 Two models of T:B collaboration .
  • Panel A illustrates the conventional model of T-B collaboration namely that the ability of T cells to recognize foreign antigen and promote immunoglobulin production depends on the recognition by TcR of peptidic antigen presented in the context of the polymophic MHC molecules.
  • TcR peptidic antigen presented in the context of the polymophic MHC molecules.
  • immunopotentiating hetercoconjugates For example, F(AB') 2 fragment of anti-CD3 are coupled to peptidic antigen to bridge the T cell with peptide specific B cells. The resulting cross-linking of the TcR by this T-B interaction would result in the localized secretion of helper factors necessary to augment B cell triggering and Ab production in the absence of an MHC-restricted interaction.
  • the body's immune system is not invincible. It succumbs to attack when it is unable to recognize the need to repel invaders, for example, when the foreign or abnormal stimulus is too weakly immunogenic to produce a response. Genetic immunodeficiency states as well as acquired immunodeficiency (e.g., drug or viral induced) also undermine the immune system's ability to respond to infection or abnormal growth such as neoplasms. Moreover, artificial suppression of the immune system, often employed in an attempt to minimize the risk of transplantation failure due to host versus graft disease, is accompanied by the risk of generating serious side. effects, e.g., infection and even tumor development. A different type of problem arises when non-selective stimulation attacks normal as well as abnormal cells, leading to self-destruction.
  • non-specific stimulation of the immune system of tumor-bearing individuals by injecting adjuvants has met with only limited success (e.g., in the case of melanoma).
  • adjuvants e.g., BCG
  • immunization with unmodified allogenic tumor cells is ineffective, and may even result in a more rapid tumor growth.
  • the present invention discloses a collection of related strategies for overcoming one or more failures of the prior art in immunotherapy.
  • the success and potential success of this strategy is demonstrated in the following examples.
  • the strategy comprises producing new immunopotentiating agents and compositions which employ, for example, the use of 1) selected immunopotentiating agents alone, 2) as "adjuvants" with second agents against which a selected response is desired, or 3) as heteroconjugates wherein the immunopotentiating agent is conjugated to the second compound.
  • FIG. 1 Shown in Figure 1 is an overview of various embodiments of the invention.
  • an immunopotentiating protein in the example shown, the immunopotentiating protein is an immunopotentiating antibody, anti-CD3 in an admixture with a peptide against which an immune response is desired.
  • the middle panel is shown a heteroconjugate embodiment, comprising a conjugate between an immunopotentiating protein (in this case an antibody) and a peptide against which an immune response is desired.
  • a bifuncitonal ligand-conjugate formed between two antibodies (e.g., anti-CD4 and anti-CD3), wherein one of the antibodies is coupled to a peptide against which an immune response is desired.
  • T cell surface molecules can activate T cells in the absence of nominal antigen (26,27).
  • the mAb apparently mimics the physiologic antigen and bypasses the T cell receptor (TcR) antigen-specific recognition mechanism.
  • TcR T cell receptor
  • T cell activation is critical to generating potent in vivo immune responses to, for example, tumor antigens, in many instances, an individual's resident T cells are not sufficient to mount an appropriate immune response.
  • the inventor has developed in vivo treatments that are capable of enhancing the immune potential of T cells, providing a powerful tool to amplify the immune system.
  • an important aspect is the ability to identify appropriate immunopotentiation agents which can be employed in immunopotentation compositions hereof.
  • This identification is routinely accomplished through the use of assays which identify potentiation and/or activation of immune system cells.
  • the inventor routinely employs assays for activation of T cells such as might be exhibited as cytolytic activity by a responder cell such as activated CD8+ cells, against a target cell such as K562 cells.
  • T cell activation can be read out by the production of lymphokines (e.g., GMCSF, IL-2, ⁇ -IFN) released by the responder cells.
  • lymphokines e.g., GMCSF, IL-2, ⁇ -IFN
  • Another useful assay employs thymidine incorporation as a measure of T cell proliferation. (65).
  • Other possible assays include IL-2 receptor expression, phosphoinositide turnover, and even Ca ++ flux (66, 67).
  • the inventor routinely employs the induction of cytolytic activity, lymphokine production and T cell proliferation as assays for immunopotentiation.
  • Exemplary assays are set forth in Tables 1 and 2 below, which were employed to identify mAb 145-2C11 (29), the mAb specific for the 25 kd protein CD3- ⁇ . The data set forth in these two tables together demonstrate the ability of the 145-2C11 mAb to activate T cells.
  • Table 1 an assay is set forth wherein the ability of the mAb to redirect lysis of cytotoxic T lymphocytes (CTLs) to irrelevant target cells is assessed. Details of the assay are as set forth in references 29 and 45.
  • the lysis was dependent on the presence of effector CTLs, as incubation of the K562 cells with the mAb alone did not result in target-cell lysis. None of several other antibodies, including anti-Lyt-2.2 and anti-LFA-l mAbs, that inhibited antigen-specific lytic activity were able to promote lysis of the K562 targets by the CTL effectors. This ability to redirect lysis of the BM10-37 CTL clone to the K562 target had in fact been used as the screening procedure for identiyfing the 145-2C11 mAb.
  • mAb 145-2C11 Activates Murine T Cells; Evidenced by Induction of Non-antigen-specific Lysis Responder E/T33:1 Stimulator E/T 10:1 % Specific Lysis Target (K562) mAb E/T 100:1 bm10 B10 None 2.8 1.7 1.5 anti-T3- ⁇ (145-2C11) 53.6 42.0 37.0 anti-Ly-6C (144-4B11) 1.0 0.6 -0.1 anti-Thy-1 (145-7E12) 1.3 -0.1 0.1 anti-Lyt-2.2 (83-12-5) 2.2 0.7 -0.6 anti-LFA-1 (H35-89.9) 0.1 0.5 0.5 YBR B10 None 3.0 2.1 2.0 anti-T3- ⁇ 44.3 34.0 23.5 anti-Ly-6C3.7 2.9 2.1 anti-Thy-13.5 0.8 1.4 anti-Lyt-2.2 1.9 0.9 1.2 anti-LFA-1 2.0 2.3 2.3 2.3
  • the recombinant human interleukin 2 was provided by Cetus (Palo Alto, CA).
  • purified T cells were cultured with 145-2C11 or control mAbs in the absence or presence of costimulating factors.
  • the 145-2C11 mAb induced significant proliferation in the absence of exogenous factors.
  • the addition of either phorbol 12-myristate 13-acetate or recombinant interleukin 2 signficantly increased the proliferative response.
  • Lyt-2 + (CD8), L3T4 - (CD4) and Lyt2 - (CD8), L3T4 + (CD4) splenic T cells proliferated in the presence of soluble anti-CD3- ⁇ mAb.
  • a soluble anti-V ⁇ 8-specific mAb has been shown to strongly stimulate CD8 T cells but to only minimally affect CD4 lymphocytes.
  • phenotypically distinct subsets of T cells might be differentially activated by antibodies specific for different components of the murine TCR-T3 complex.
  • Table 2 mAb 145-2C11 Activates T cells as Evidenced by Induction of T-Cell Proliferation Exp.
  • t spleen cells were incubated with antibodies to the CD8 or CD4 molecules followed by rabbit complement (C'). In both cases, antibody treatment resulted in the elimination of > 95% of the corresponding cell population, as judged by flow cytometry.
  • C' rabbit complement
  • Phorbol 12-myristate 13-acetate (10 ng/ml).
  • Recombinant interleukin 2 50 units/ml).
  • antibodies against CD3- ⁇ have the ability to provide immunopotentiation in the forgoing assays, as measured by proliferation, secretion of lymphokines and the expression of interleukin 2 receptor (IL-2R).
  • IL-2R interleukin 2 receptor
  • Similar phenomena occur following the treatment of mice with doses of, e.g., on the order of 4 ⁇ g to 400 ⁇ g anti-CD3 mAb in vivo (30; Figures 2-4).
  • anti-CD3 mAbs also cause TcR coating, modulation and depletion of T cells from peripheral blood and lymphoid organs.
  • the net result of treatment with high doses of anti-CD3 mAbs in vivo is immunosuppression.
  • the present invention is concerned with compositions and protocols which are tailored to allow /the administration of relatively lower doses.
  • assays such as those set forth above for T cell activation and potentiation can be employed in a variety of fashions to assist in achieving the goals of the present invention.
  • assays such as these can be employed for screening hybridoma colonies to identify those which secrete mAbs having the desired immunopotentiating effect.
  • assays such as these can be readily employed to screen for and identify other suitable immunopotentiation agents such as immunopotentiating bacterial or mycoplasmal proteins.
  • assays such as these can be employed as an initial step in the determination of appropriate dosages in test animals (e.g., in terms of mg/kg body weight) which will provide immunopotentiation as opposed to immunosuppression.
  • anti-CD3 administered to humans during a clinical trial on the use of anti-CD3 in cancer treatment, is found to act in similar fashion to activate T cells as in the above test systems ( Figures 5 A and B).
  • antibodies or reagents specifically directed at T cell subsets are even more useful as immune adjuvants without producing associated immunosuppression, because this specificity ensures that the majority of T cells will remain unaffected.
  • the potentiation reagents used will in certain cases activate all T cells, and in others subsets of T cells will be selectively activated.
  • T cell subsets are defined as individual families of T cells that share common features including cell surface proteins such as CD4 and CD8 which are expressed on 66% and 33% of the T cells, respectively, Ly-6 and CD28 expressed on distinct families of T cells, or the TcR proteins. (11, 15, 16)
  • T cell Epitopes Proteins in the T cell Receptor Complex (TcR) a. Non-polymorphic (Monomorphic) Epitopes (e.g. CD3- ⁇ , ⁇ , ⁇ , ⁇ or epitopes from the constant region of the TcR ⁇ , ⁇ , ⁇ , ⁇ chains) b. Polymorphic Subset Specific Epitopes (e.g. epitopes expressed on the variable portion of TcR, V ⁇ , V ⁇ V ⁇ or V ⁇ chains) 2. Proteins Not Associated with the TcR a. Non-polymorphic (Monomorphic) Epitopes (e.g. Thy-1, CD2) b. Polymorphic Subset Specific Epitopes (e.g. CD4, CD8, Ly-6, CD28)
  • TcR T cell Receptor Complex
  • SEB Staphylococcus enterotoxin
  • Figs. 7-10 The response of the immune system to Staphylococcus enterotoxin (SEB) as an immunopotentiating agent is surprisingly stronger than, although qualitively similar to, that seen in response to anti-CD3.
  • SEB produced a dose responsive increase in allogeneic cell proliferation in mice (Fig. 7).
  • SEB administration exhibited a surprisingly enhanced IL-2R expression by T cells, and also showed a dose response.
  • Fig. 8 proliferative effects evoked by SEB administration, as measured in the presence of RIL-2, were also of a surprising magnitude (Fig. 9).
  • V ⁇ 8 + cells are a subset of T cells representing only 20%, and are important because they mediate a variety of immune responses.
  • SEB to preferentially expand V ⁇ 8 + is of particular significance because it activates only a small subset of polyclonal T cells without directly affecting the majority of the T cell response.
  • T cells may play a critical role in engraftment.
  • T cells may be required for limiting cytomegalovirus and Epstein-Barr virus infection in transplanted individuals.
  • specific T cells may provide potent graft vs. leukemia responses that eliminate residual tumor.
  • T cells may provide a critical role in engraftment by producing a variety of growth factors such as GM-CSF.
  • aspects of the present invention may be useful in addressing this problem.
  • immunosuppression and immunopotentiation The use of certain doses of activating anti-CD3 antibodies in vivo will not only prevent GvHD but will also activate endogenous T cells to produce hematopoietic growth factors that facilitate engraftment. Exemplary doses for achieving this effect will range from 5 mg/kg to 20 mg/kg, with about 10 mg/kg being proposed as optimal.
  • the ability of immunopotentiating agents to increase hematopoesis is demonstrated, e.g., by studies wherein it is shown that specific recruitment of stem cells such as myelocyte and bands occurs upon administration of such agents in humans, and increased colony formation in both mice and humans.
  • mAbs directed at activation antigens present on stem cells or other hematopoietic cells may induce factor production or cell differentiation that will facilitate engraftment in the absence of T cells.
  • mice were first treated i.p. with 250 ⁇ g of the anti-CD3 mAb (145-2C11) and then exposed to 500 rads gamma-irradiation. Within one to two hours after irradiation, the mice were injected with spleen cells from a C57BL/10 mouse. Control mice, not pretreated with anti-CD3 died of GVH disease within 2 weeks. In contrast, 75% of mice given a single dose of anti-CD3 survived.
  • the anti-CD3 antibody was preadministered because it is proposed that circulating anti-CD3 antibody would modulate TcR from host T cells and thereby inhibit HvD reactions in experiments focused on enhancement of allogeneic bone marrow engraftment.
  • circulating anti-CD3 mAb depleted alloreactive T cells from the donor innoculum before GvH reactions can be initiated.
  • the mAb effects were examined by CSF assays, in vivo , and CFU-C, BFU-E and CSF assays in vitro .
  • mAbs may be directed to recognize activation molecules expressed on hematopoietic cells.
  • Ly-6A also known as Scal
  • Ly-6C is expressed on the earliest hematopoietic stem cells and studies have shown that mAbs directed at the Ly-6A epitope will activate T cells.
  • a second mAb 143-4-2 which reacts with the Ly-6C epitope expressed on 40% of non-stem cells in the bone marrow has also been shown to activate T cells [27].
  • Table 4 presents data demonstrating the in vivo effects of anti-CD3 on hematopoesis.
  • the studies shown in Table 4 were performed by injecting 250 ⁇ g of anti-CD3 on day 0. Bone marrow cells were harvested on days 4 and 10 and examined in vitro for bone marrow colony formations as above. As can be seen, the administration of anti-CD3 to mice in all cases resulted in profound increases in hematopoetic progenitor cell activity.
  • an important aspect of the invention is the recognition that immunopotentiation agents described herein can be employed an "immunoadjuvants" in order to evoke an improved immune response to compounds against which such a response is desired.
  • immunopotentation agents may be formulated with or otherwise coadministered together with such compounds in order to improve or to develop an immune response against such compound.
  • Various studies have been performed which serve to demonstrate the surprising potential for the immunopotentiation agents hereof to act in this manner as immunoadjuvants.
  • KLH Keyhole Limpet Hemocyanin
  • the adjuvants studied were PBS (phosphate buffered saline control), CFA (complete Freund's adjuvant) and anti-CD3.
  • the study was carried out as follows: 100 ⁇ g of KLH was injected on day 0 i.p. in the presence or absence of adjuvant. Mice were boosted on day 14 and bled at weekly intervals. Antibody activity was assessed in sera by standard ELISA.
  • mice were tested for their ability to produce specific antibodies in response to challenge by various routes with a KLH-linked peptide, termed peptide 1005/45-KLH, with and without co-administration of anti-CD3.
  • This peptide is derived from the CD4-binding portion of the HIV-GP120 molecule.
  • Mice were administered the test immunogen, 1005/45-KLH, either 100 ug subcutaneously (sc) or 100 ug intraperitoneally (i.p.) at day 0 and 14, followed by administration of 4 ug of anti-CD3 (145-2C11) i.p. in test animals.
  • One object of this invention was to directly couple a peptide to an immunopotentiating protein, e.g., monoclonal antibody or bacterial enterotoxin, to provide a more potent immunogen than a peptide alone.
  • an immunopotentiating protein e.g., monoclonal antibody or bacterial enterotoxin
  • What has been achieved in this invention is to develop a "pass key" which tricks the cell into thinking it sees a foreign antigen being presented, causing it to be activated.
  • the T cell activity is directed towards the unique antigen-specific B cell target.
  • the second protein is isolated and purified by standard methods from diseased or abnormal tissue, from an infectious agent, or by genetic engineering a specific amino acid sequence using standard molecular biology techniques.
  • Embodiments of second proteins in general are shown in Table 6.
  • a specific embodiment would be the amino acid sequence, T1, encoded within the gp 120 protein of the HIV virus: K Q I I N M W Q E V G K A M Y A.
  • Table 6 Embodiments of the second protein * in the heteroconjugate Category Size Example Hapten Small Chemical Compounds FITC ( Figure 7,8) Peptide 8-50aa Peptide 18 Protein >50aa gp 120 (HIV Virus) *Includes an epitope against which a humoral immune response is desired.
  • cross-links are formed between the immunopotentiating protein and the second protein by use of any of the methods which link proteins to form bonds that are stable under normal physiologic conditions.
  • methods include biotin-avidin bridges, SPDP functional groups, and cross-linking of maleimide and SH groups.
  • This invention is also directed toward a method of stimulating an immune response in persons identified who are in need of such stimulation due to having diseases or infections.
  • the heteroconjugate is prepared with a pharmaceutically acceptable excipient or diluent to form a therapeutically effective compound.
  • Persons who are candidates for this treatment include those who have a tumor, are immunocompromised or have contracted AIDS or other viral, bacterial, fungal, or parasitic infections.
  • Another embodiment is to stimulate the immune response by administering only the immunopotentiating protein of the heteroconjugate not linked to the second protein.
  • the immunopotentiating protein may be administered before, after or in conjunction with the second protein.
  • a single immunopotentiating agent may be used.
  • the monoclonal antibody which is specific for a non-polymorphic or polymorphic T cell surface protein is administered to the persons by intravenous route.
  • the monoclonal antibody must not be immunosuppressive nor adversely affect CD3/TcR expression at the dose used.
  • One embodiment disclosed is to use monoclonal antibody against the CD3 cell surface antigens associated with the TcR.
  • the mAb mimics a physiologic antigen and bypasses the TcR antigen - specific recognition mechanism.
  • the epitope is a tumor
  • Methods disclosed in this invention enhance the immune potential of T-cells and other components of the immune system and provide powerful tools to amplify the immune system.
  • An object of the invention is to augment the effects on the immune system of weakly immunogenic small peptides or proteins. For example, conjugating proteins to monoclonal antibodies produces a stronger response by bypassing the requirements of immunogenicity mandated by MHC restriction and antigen recognition by T cell receptor.
  • the strategy of this invention is to stimulate T cell immune reactivity using mAbs.
  • T cell activation enhances the ability of an individual to reject a malignant tumor in a specific manner and produces long lasting tumor immunity. Therefore, the antigen-like effect of monoclonal antibodies to TcR/CD3 structures are disclosed as a means to specifically enhance immune function in vivo in tumor bearing individuals.
  • This invention allows for the direct interaction of the vital components of the immune response in developing strong cellular and humoral immunity. It is directed also toward protecting those who are not yet affected with the disease or condition by disclosing a vaccine comprising the immunogenic heteroconjugate described above.
  • the use of this heteroconjugate does not require the interaction of the peptide with the major histocompatibility complex antigens, which presents a very significant advantage, that is, bypassing the polymorphic response which is usually a problem with the MHC system. All immune responses are dependent on the ability of T cells to recognize processed antigen associated with major histocompatibility antigens (MHC).
  • MHC major histocompatibility antigens
  • Any vaccine approach which utilizes HIV peptides or inactivated virus antigen must depend on the ability of antigenic peptides to bind the appropriate MHC antigens necessary to initiate an immune response.
  • the tremendous polymorphism of the MHC antigens expressed in the population and the variation of the virus, developing a successful HIV vaccine for general use faces major obstacles.
  • developing an ideal form of immunotherapy and vaccines against, for example, AIDS could be achieved by increasing the antigenicity of the peptide MHC interaction or boosting the activity of the immune cells, thus reducing the threshold of antigenicity necessary to trigger specific immune responses and memory following antigen exposure.
  • a more general vaccine is created by use of the heteroconjugate which is an aspect of this invention because the extensive genetic diversity of the MHC is not a factor in binding to T cells.
  • the number of peptides that are present in sufficient quantity to bind both the MHC and TcR, thus inducing an immune response is small, factors limiting the success of vaccines.
  • vaccines are comprised of heteroconjugates.
  • Monoclonal antibodies which are used as the immunopotentiating protein in the vaccine include T-cell surface antibodies directed against non-polymorphic or polymorphic T-cell surface molecules.
  • the second protein which is used as the other portion of the heteroconjugate includes those which are not capable of stimulating the immune system sufficiently to achieve immunization unless they are administered in a conjugate with the monoclonal antibody.
  • An object of this invention is an approach to therapy which activates the host antitumor cellular effector mechanisms, even though the epitope is only weakly immunogenic and could evade host recognition and rejection unless augmented.
  • Major forms of immuno-deficiency may be due to the inability of antigen to trigger a primary immune response due to suboptimal antigen presentation and T cell activation.
  • compositions for stimulating immunity In addition to the administration of compositions for stimulating immunity, the present invention also contemplates that certain immunological products which are produced as the result of such an administration may provide a benefit to some individuals, particularly immunocompromised individuals. That is, it is contemplated that the immunopotentiating compositions of the present invention may be employed to produce antibody compositions, such as gamma globulin fractions, which may be administered to individuals for the development of passive immunity.
  • immunopotentiating compositions hereof which contain appropriate compounds against which an immune response is desired, whether such compositions are in the form of "adjuvant" compositions or heteroconjugates, are administered to disease-free individuals in an amount effective to elicit a specific immune response against the second compound.
  • the resultant gamma globulin fraction is then obtained and purified by well known techniques, and administered in effective amounts to individuals in need of such treatment.
  • the present invention contemplates that other immunological products, such as activated T and B cells will also be useful for such purposes, e.g., in the treatment of cancers and even HIV infections.
  • the development of the invention required production of the immunopotentiating agents, testing their effects in vitro , and finally developing their in vivo effects in mice as models, and in humans for purposes of treatment.
  • in vivo effects disclosed in this invention are not absolutely predictable from the in vitro effects because an intact organism's response reflects interactions of a complex immune system, whereas, in vitro , individual components can be controlled, and system interactions are difficult to simulate. In vivo , various components which reacted separately in the laboratory in certain fashions, may not interact appropriately when their actions are combined.
  • Monoclonal antibodies were generated which are reactive with the T cell surface structures expressed as alloreactive cytotoxic T cell (CTL) clones and involved in T cell activation.
  • CTL cytotoxic T cell
  • MAbs specific for these cell surface molecules were identified using an assay (developed by Bluestone, the redirected lysis assay (33), see also Example 13) based on the ability of antibodies reactive with the TcR complex to induce antigen-specific CTL to lyse cells which are not their natural targets. This activity depends on the ability of the antibody to both activate the CTL via the TcR and multivalently crosslink the effector to the target cells via the Fc receptor (FcR) on the target cells.
  • Fc receptor Fc receptor
  • the mAb, 145-2C11 was derived by fusing spleen cells from an Armenian hamster immunized with a murine CTL clone to SP2/0 cells, and screening the resulting hybridoma supernatants in the redirected lysis assay. This mAb immunoprecipitated the complete TcR complex using non-ionic detergents but reacted specifically with a 25-kD protein component (CD3- ⁇ ) of the antigen-specific TcR complex. 145-2C11 mAb may also be obtained by-growing hybridoma cells in an Acusyst P machine (Endotronics, Minn., MN) and then collecting the supernatant. Antibodies are then purified by 50% ammonium sulfate precipitation followed by gel filtration on an ACA 34 Ultragel column [BF Biotechnics, Savage, MR]
  • mAb 143-4-2 was derived by fusing spleen cells from a BALB/c mouse immunized with a murine CTL clone and screened as in Example 13. This mAb defines a novel cell surface molecule involved in T cell activation. The expression of the 143-4-2 defined epitope was expressed on the Ly-6C molecule and was restricted in its lymphoid expression to bone marrow cells and to a subset of peripheral CD8+ cells (34).
  • the anti-Ly-6.2C antibody can promote the lysis of target cells that do not bear antigens by alloreactive CTL clones and, in the presence of cofactors (PMA or IL-2), induced a subset of CD8+ cells to proliferate, perhaps through an autocrine pathway.
  • PMA or IL-2 cofactors
  • the antibody described has antigen-like effects as described for anti-TcR complex reagents, it was shown that the Ly-6.2C molecule was not associated on the cell surface with components of the TcR complex.
  • mAbs were purified by ion exchange chromatography and gel filtration.
  • F(ab')2 and Fab fragments were prepared by pepsin and papain cleavage of purified antibody, respectively (36).
  • Additional mAbs were prepared that can be employed as immunopotentiating proteins in vivo to activate T cell subsets and hematopoietic stem cells. These include: UC3-10A6 and UC7-13D5 mAbs which are specific for the TcR ⁇ ⁇ receptor; UC3-7B7 which is specific for Thy-1; 145-4B11 and D7 (obtained from Tom Malek, Miami, FL) which are specific for Ly-6A (also known as the Scal antigen expressed on bone marrow stem cells); and H597.57, an anti-TcR ⁇ mAb (Ralph Kubo, Denver, CO).
  • Immunopotentiating proteins disclosed in this invention comprise monoclonal antibodies prepared as follows:
  • Any microbial component e.g. a bacterial enterotoxin, may be assayed for T cell activption.
  • Enterotoxins may by purchased from biochemical suppliers.
  • Toxins that satisfy criteria of activation assays may be subjected to standard protein purification techniques, gel filtration and exchange chromatography.
  • Preferred epitopes may be selected from those shown in Table 3.
  • mice were given different doses and their lymph nodes and spleen cells were examined for IL-2R expression by flow-cytometry. IL-2R expression was enhanced at the three doses tested (4, 40 and 400 micrograms) and plateaued at 400 micrograms. (Fig. 2). When the same lymphoid cells were incubated in media containing human rIL2,their proliferation was enhanced in proportion to their IL-2R expression.
  • the immune suppression which results from a dose of 400 microgram of anti-CD3 was the result of T cell depletion, T cell receptor blockade and modulation of the TcR complex.
  • the purpose of this example was to test the effect of mAb treatment on infection.
  • Administration of low doses of anti-CD3 prevented the lethal pneumonia caused by the Sendai virus in >60% of mice.
  • Anti-CD3 treated, virally-infected mice also developed lasting virus-specific immunity as evidenced by their ability to withstand a subsequent dose of Sendai virus of 1000 times the LD 50 dose.
  • Treated mice also developed a Sendai virus specific DTH and antibody response similar to mice immunized with a non-virulent Sendai virus vaccine.
  • the 129/J strain of mice were also protected by the anti-CD3 treatment. Because virus susceptibility in these mice has been shown to be caused by an inadequate generation of NK cell responses, it appears as if NK activity induced by the mAb treatment contributes to viral protection.
  • the purpose of this example was to determine the feasibility of immunotherapy designed to prevent tumor outgrowth and induce tumor immunity by administering anti-CD3 in vivo to activate T cells. This possibility was tested in mice with the eventual objective of pursuing similar strategies in humans.
  • mice Effects of the anti-CD3 treatment on malignant tumor growth in vivo were tested in mice.
  • the C3H fibrosarcoma 1591-Pro-4L a weakly immunogenic ultraviolet-light-induced murine tumor that lacks cell surface CD3 and FcR does not react directly with the anti CD3 used for treatment.
  • This malignant tumor grows progressively in 95 percent of normal CD3H mice and eventually kills the mice by infiltrative growth without macroscopic evidence of metastases. None of the mice treated with 4 ⁇ g of anti CD3 developed tumors in this experiment. (Table 7).
  • Immunopotentiating effects include increased tumor specific T cells, lymphokine-activated killer cell activity, increases in tumor necrosis factor (TNF) detected in the serum of treated, not control mice.
  • Findings reported in Table 7 were confirmed using a metastatic tumor system MCA102. Lung metastases in mice having established MCA102 tumors are significantly reduced by the mAb treatment. In one experiment, mice injected with tumor alone developed a mean of 105 ⁇ 26 lung metastases while mice treated with anti-CD3 mAb 3 or 10 days after tumor inoculation developed 25 ⁇ 6 and 44 ⁇ 14 lung metastases respectively.
  • Table 7 Summary of Tumor Incidence at Day 28 in Anti-CD3- Treated Mice There was no recurrence, late outgrowth, or tumor regression after 28 days.
  • SE Staphylococcal enterotoxins
  • Tables 8a and 8b Results of studies with SEB are presented in Tables 8a and 8b below.
  • Table 8a in vivo administration of SEB results in a selective activation of V ⁇ 8 + T cells.
  • Table 8b demonstrate that while two out of six mice treated with four ⁇ g of anti-CD3 developed tumors following tumor innoculation, the administration of 50 ⁇ g of SEB resulted in no incidence of tumor development.
  • Table 8a IL-2 Receptor Expression on T Cells from SEB Treated Mice.
  • C3H mice were treated either with anti-CD3 (145-2C11) or with one of three doses of Staphylococcus enterotoxin (5 ⁇ g, 50 ⁇ g, 500 ⁇ g). There were untreated controls. Both syngeneic and allogeneic responses were determined by 3 H uptake of lymphocytes. As shown in Figure 7 treatment with this single agent promoted an allogeneic response. A clear dose effect was also observed.
  • mice treated with SEB showed a significantly lower tumor incidence (Table 8b).
  • Fig. 8 presents results of SEB treatment of mice in vivo , in which IL-2R expression showed a clear dose response effect. Specific and preferential stimulation and expansion of V ⁇ 8+ cells in the SEB-treated mice is further shown in Fig. 10.
  • Figures 5A and B, 11, 12 illustrate the response of human cells to low doses of OKT3. Different lymphocyte subsets show a similar pattern of response, but different absolute percentages of total lymphocytes. These results are from the ongoing clinical trial of OKT3 in treating patients with cancer. The methods for this trial are described in Example 12.
  • anti-CD3 will depend on the clinical condition. Depending on this, doses are selected. Mice were treated with 500 R radiation to suppress their immune response thereby facilitating transplantation. After 13 days, however, the graft was rejected in control animals. Dramatic increased survival of 75% was observed at the same point in time for animals treated with 250 ⁇ g of anti-CD3, and 30% survival after 25 ⁇ g of anti-CD3. (Fig. 13). This effect is believed due to induced hematopoesis which revitalizes the graft.
  • Example 9 Use of a Hapten or Peptide as a Second Protein in a Heteroconjugate or as a Combination Administered Concurrently
  • Fluorescein isothiocyanate Sigma
  • DNP Dinitrophenol
  • Examples of other second proteins are synthetic peptides of HIV, 13-23 residues long prepared by the multiple simultaneous peptide method of solid-phase synthesis in polypropylene mesh. These are coupled to the anti-CD3, BSA OVA or KLH using Maleimide as described in Example 2C.
  • vSC-8 recombinant Vaccinia vector containing the bacterial LACz gene
  • vSC-25 recombinant Vaccinia vector expressing the HIV env glycoprotein gp160 of the HTLV III b isolate of HIV structural or regulatory proteins
  • mice are conventionally immunized with 100 ⁇ g HIV/pep-KLH, or HIV/pep in Complete Freund's Adjuvant (Difco Laboratories) intraperitoneally 1-12 months before use either concomitantly or after injection of 4 ⁇ g of anti-CD3 mAb.
  • mice may be immunized with anti-CD3 coupled FITC or HIV peptide.
  • mice are low responders to OVA, depending on their MHC haplotype.
  • the low response was due to the inability of carrier-specific T cells to recognize OVA peptides in the context of self MHC.
  • Anti-CD3 is coupled to various OVA peptides including amino acids 323-334 which is known to be immunogenic in H-2 d strains as well as other peptides not capable of generating an immune response (46). These experiments were performed to assay the ability of anti-CD3/hapten heteroconjugates to induce immune responses in low responder haplotypes. In particular, the activation of B cells by a direct crosslinking of T and B cell by the heteroconjugate is believed to bypass the requirement for antigen presentation by MHC molecules (Figure 17). Thus, these model antigens provides the basis for initial analyses of the immune regulation in anti-CD3-treated mice.
  • the UV-induced skin tumor lines 1591-Pro-4-L, 6139-Pro-1, 1316-Pro-1 and 1591-Var4L were produced as described (50). Tumor cells were passed by serial culture in complete media. For in vivo studies, tumor cells were injected into C3H nude mice. Solid tumors isolated 3 weeks later were cut into 2 mm fragments, and 10 of these fragments were injected into a single subcutaneous location using a 14 gauge, 2 inch long trochar needle. Tumor presence and size were determined at one month in mice. YAC-1 T lymphoma cells and P815 mastocytoma cells were used as targets in some assays in vitro .
  • Specific antigens may be prepared from tumor cells isolated by serological techniques or suspension dilutions. These antigens were purified by standard methods, for example, density gradient centrifugation or velocity immunosedimentation. The identity of the antigen was confirmed by serology or histological examination. Cells are lysed and used in the assays described in Example 3 to identify unique tumor antigens. Protein extracts are prepared by standard methods including molecular sizing and ion exchange chromatography. Further molecular and biochemical analysis for T specific antigens may be performed by established cloning techniques.
  • mice between 8 to 12 weeks of age were obtained from The Jackson Laboratory, Bar Harbor, ME.
  • a thymic BALB/c mice and NIH Swiss mice were obtained from the National Institutes of Health small animal production facility.
  • Example 13 Administration of the Heteroconjugate or Immunopotentiating Antibody to Patients
  • FIG. 5 A and B In vivo response to OKT3 is shown in Figures 5 A and B, 11, 12.
  • Patients selected for treatment were those who have histologically documented malignancy which is either evaluable or measurable and for which standard therapy is unavailable. These patients should not have had prior administration of murine antibodies or history of documented or suspected life threatening immune mediated disorders such as asthma. There should not be concurrent drug therapy or infection. Patients should not be pregnant or have either class III or IV cardiac disease. (New York Heart Association Classification)
  • the monoclonal antibody to be administered in a preferred embodiment was Muromonab-CD3 (OKT3), a murine monoclonal antibody directed against the T3 (CD3) antigen of human T cells.
  • This antibody reacts with a 20,000 dalton molecule (CD3) in the membrane of human T cells that has been associated in vitro with the antigen recognition structure of T cells, and which is essential for signal transduction.
  • CD3 20,000 dalton molecule
  • anti-CD3 reacts with most peripheral blood T cells and T cells in body tissues but has not been found to react with other hematopoietic elements or other body tissues. It is a potent mitogen in vivo .
  • the drug was administered through a needle in the contralateral vein by intravenous infusion.
  • Monomurab-CD3 was pushed over 30 to 60 seconds using a 20 cc syringe containing OKT3 dose in 5% human serum albumin.
  • the patient's vital signs were monitored every five minutes for the first fifteen minutes, then at two hours and four hours until twenty-four hour post infusion. Patients received 3 treatments at 2 week intervals unless precluded by toxicity. Toxic response is defined by published criteria.
  • Orthoclone OKT3 (Muromonab-CD3) sterile solution is a murine monoclonal antibody to the T3 (CD3) antigen of human T cells which functions as an immunosuppressant.
  • the antibody is a biochemically purified IgG2 immunoglobulin with a heavy chain of approximately 50,000 daltons and a light chain of approximately 25,000 daltons.. It is directed to a glycoprotein with a molecular weight of 20,000 in the human T cell surface which is essential for T cell function. Because it is a monoclonal antibody preparation, Orthoclone OKT3 sterile solution is a homogenous reproducible antibody product with consistent measurable reactivity to human T cells.
  • Each 5 ml ampule of Orthoclone OKT3 sterile solution contains 5 ml (1 milligram per ml) of Muromonab-CD3 in a clear colorless solution which may contain a few fine translucent protein particles.
  • Each ampule contains a buffer solution (Ph 7.0 ⁇ 0.5) of monobasic sodium phosphate (2.5ml), sodium sulphate (9.0 mg), sodium chloride (43 mg), and polysorbate 80 (1.0mg) in water for injection.
  • the proper name Monomurab CD3 is derived from the descriptive "murine monoclonal antibody.”
  • the CD3 designation identifies the specificity of the antibody as the cell differentiation (CD) cluster 3 defined by the International Workshop on Leukocyte Differentiation Antigens.
  • Orthoclone OKT3 is supplied as a sterile solution and packed in 5ml ampules containing 5 ml of Muromonab-CD3. These vials are stored, refrigerated at 2-8°C. Prior to administration the Orthoclone OKT3 protein solution may develop a few fine translucent particles. These have been shown not to affect its potency. The product is prepared for injection by drawing the solution from the vial into a syringe through a low protein binding 0.2 micrometer filter. The filter is discarded and the needle attached for IV bolus injection. Orthoclone OKT3 is delivered as an IV bolus in less than one minute. This drug should not be administered as an intravenous infusion or in conjunction with other drug solutions. When necessary, Orthoclone OKT3 solution may be diluted with sterile saline.
  • OKT3 For the preparation of OKT3 for injection, the antibody was drawn into a syringe as described above. The appropriate dose of OKT3 was then added to 15 to 20cc 5% human serum albumin in normal saline and placed in a 20cc syringe. Toxicity was monitored and may include fever and chills, shortness of breath, allergic reaction, chest pains, vomiting, wheezing, nausea, diarrhea or tremors.
  • LAK Lymphokine activated killer cells
  • Effector cells and 51 Cr-labeled target cells were added to wells of 96-well tissue culture Seroclusters (Costar, Cambridge, MA) at various effector to target ratios, in triplicate, in a total volume of 200 ⁇ l of complete medium.
  • the monoclonal anti-TcR antibody, 2.4G2 (47) was added to wells at a concentration of 10% cs where indicated. Plates were then incubated for 4 or 6 hours at 37C° following which 100 ⁇ l of supernatant was aspirated and analyzed on a gamma counter (Micromedic Systems, Inc., Horsham, PA).
  • E cpm which represents the 51 Cr released from the target cells incubated with effector cells
  • T cpm represents release of radiolabel from target cells in a 0.05N solution of HCL
  • S cpm represents background release of target cells cultured in media in the absence of effector cells.
  • the data should represent at least three experiments with identical results. Standard errors for all 51 Cr release values should be less than 5%.
  • Virus-specific CTL populations were generated in vitro using 5x10 6 splenic responder cells mixed with 2.5x10 6 irradiated (3300 rad) Vaccinia virus infected syngeneic spleen cells (1 hr., 37°C, multiplicity of injection, 10:1) in 2ml cultures in complete media (RPMI 1640 supplemented with 10% selected fetal, calf serum (FCS), sodium pyruvate, nonessential amino acids, glutamine, and 2-mercaptoethanol). Cytolytic activity is assayed after 6 days on appropriate virally-infected targets or peptide-pulsed syngeneic or allogeneic 51 Cr-labeled targets.
  • T cells were cultured in RPMI 1640 medium containing 10% FCS and 2-mercaptoethanol, in the presence or absence of mAbs and/or factors in flat-bottomed microliter 96 wells plates.
  • purified anti-CD3 mAb were immobilized on plates as a means of activating cells in the absence of Fc receptor + cells.
  • the cells were pulsed with 1 ⁇ Ci [ 3 H] thymidine for 16 hours, the samples were harvested and incorporation of radioactive isotope measured using a scintillation counter.
  • the secretion of soluble lymphokines were monitored by the ability of culture supernatants of activated T cells to support the growth of the lymphokine-dependent HT-2 cell line. 5x10 3 HT-2 cells were cultured for 24-36 hours with the supernatant to be assayed, then pulsed for 16 hours with 1 ⁇ Ci [ 3 H] thymidine.
  • This protocol is adaptable in time and cell number when assayed in humans versus animals.
  • Antibody responses were assayed in vitro by two methods: (1) Plaque forming cells (PFC) responses were measured on the day of culture by assaying TNP, HIV peptide of FITC-conjugated sheep erythrocytes as described (48). Indirect IgG responses were evaluated by blocking the IgM plaques with goat-anti-mouse IgM and developing with a rabbit anti-mouse IgG. (2) Direct binding enzyme-linked immunosorbant assays (ELISA) of FITC, TNP or peptide binding antibodies were carried out as follows.
  • Nunc ELISA plates were coated overnight with 100 ⁇ l of 50 ⁇ g/ml FITC-BSA, TNP-BSA or HIV-peptide-BSA in Voller's buffer (4 ° C) Plates were washed extensively with 0.05% Tween 20 diluted in PBS. Free sites were blocked by incubating the plates with 0.5% BSA for 1 hr. at room temperature. One hundred ⁇ l of control or immune sera, diluted in PBS-Tween was added to each well in 3-fold serial dilutions and incubated for 1 hr. After washing, 100 ⁇ l of 1 ⁇ g/well horseradish peroxidase conjugated goat anti-mouse IgG were added.
  • Cell surface-labeling with 125 I was performed by the lactoperoxidase method as described previously (52). Labeled cells were lysed in 1% digitonin buffer, and precleared with hyperimmune rabbit serum. Lysates were immuno-precipitated with anti-CD3 (145-2C11) and protein A agarose beads. Immunoprecipitates were eluted from the protein A agarose in non-reducing sample buffer (containing 2% SDS) and were either directly electrophoresed or reimmunoprecipitated with specific anti-TcR sera prior electrophoresis. The generation of the anti-TcR antisera has been described previously (53).
  • Example 16 Coupling the Immunopotentiating Protein to the Second Protein to form a Heteroconjugate
  • any of the various procedures available to form cross-links between proteins can be utilized.
  • the method chosen must not destroy the immunopotentiating activity of the heteroconjugate.
  • both of the proteins to be linked are individually coupled to biotin molecules.
  • Avidin then forms a bridge between the biotin molecules, thereby linking the two proteins. This is not a covalent bonding.
  • a variation of the biotin-avidin method is to couple one protein to biotin, the other to avidin, and to mix the two compounds resulting in biotin-avidin links.
  • Disulfide groups are introduced into one of the proteins to be conjugated, and thiol groups are introduced in the other by pyridyldithiolation and subsequent reduction with dithiothreitol. Upon being mixed, the two substituted proteins conjugate by means of a disulfide bond. The details of this method are provided by Carlsson (42). This method has been successfully applied to form antibody-toxin conjugates (43).
  • SH groups on an one protein are fully alkytated with 0-phenylenedimaleimide to provide free maleimide groups.
  • the other protein is reduced to produce SH groups.
  • the two preparations are then cross-linked (44).
  • PROCEDURE
  • PROCEDURE
  • Example 17 Cell separation and tissue culture techniques
  • MHC major histocompatibility antigens
  • the efficacy of vaccines in particular those which may be weakly immunogenic, may be improved by modifying the foreign antigen such that it is more immunogenic, or allowing the use of peptides which are not immunogenic under normal conditions, since they would not bind MHC, but which may constitute a conserved site on the major HIV viral proteins.
  • mice are immunized with an amount of heteroconjugate between the amounts of 100ug and 5mg added to 30 ⁇ l-1ml of a physiologic salt solution or Freund's adjuvant (Difco). Alum or pertussis may be added as adjuvants.
  • This solution is then injected into a mammal (mouse, rat, hamster, rabbit) or human to stimulate an immune response against the second protein. Injection may be subcutaneous, or by an intravenous or intramuscular route. Preferred sites for mice are the foot pad or the base of the tail. A booster shot is given 30 days after the primary injection.
  • delayed skin hypersensitivity or direct tests for antibodies to the amino acid sequence in the second protein of the heteroconjugate may be performed.
  • Synthetic peptides corresponding to selected sites are prepared using standard methods of solid-phase peptide synthesis on a Vega 250 peptide synthesizer using double dicyclohexylcarbodimide-mediated couplings (57,58) and butyloxycarbonyi (Boc)-protected amino acid derivatives. Hydroxybenzotriazole preactivation couplings are performed when coupling glutamine or asparagine. The extent of coupling is monitored using the qualitative ninhydrin test and recoupling is performed when ⁇ 99.4% coupling is observed. Peptides are cleaved from the resin using the low/high hydrogen fluoride (HF) method (59).
  • HF low/high hydrogen fluoride
  • peptide env T2 For peptide env T2, standard HF cleavage is employed as removal of the tryptophan formyl protecting group is found not to be required for antigenic activity. Peptides are purified to homogeneity by gel filtration and reverse phase HPLC (60). Composition was confirmed and concentration determined by amino acid analysis (61).
  • native gp120 is purified from virus-infected cells as described (62).
  • the recombination proteins R10 and PB1 are produced by cloning restriction fragments Kpn I (nucleotide 5923) to Bgl II (nucleotide 7197) or Pvu II (nucleotide 6659) t o Bgl II (nucleotide 7197) from the BH10 clone of type III human T-cell lymphotropic virus (HTLV-III g ) into the Repugen expression vector, followed by expression in Escherichia coli and purification as described (63).
  • R10 is initially solubilized in 20 mM Tris-HCl, pH 8.0, with 10 mM 2-mercaptoethanol at 0.22-0.66 mg/mi.
  • B1 is solubilized in 8 M urea at 1.9 mg/ml.
  • Protein R10 represents residues 49-474 of the HTLV-III g envelope protein with 25 non-HTLV-III vector-derived residues at the N terminus and 440 such residues at the C terminus.
  • Protein PB1 represents residues 294-474 of gp120 with 30 and 24 non-HTLV-III residues at the N and C termini, respectively.
  • the N-terminal residues of PB1 are partially shared with those of R10, whereas the C-terminal residues are unrelated.
  • Example 21 Models to Test Use of Anti-CD3 as an Immunopotentiating Agent in Anti-Viral and Anti-Tumor Immunity
  • a preclinical huSCID model for an anti-human CD3 and staphylococcus enterotoxin augmented viral and tumor immunity may be developed as a model for the human counterpart, OKT3, or SEB to activate immunity in a similar manner.
  • serum from patients have been shown to contain soon after OKT3 injection various lymphokines and cytokines including TNF, ⁇ -interferon, IL-2, and GM-CSF. Therefore, like anti-murine CD3, OKT3 activates T cells in vivo .
  • a model may be developed to examine the OKT3 antibody.
  • SCID mice populated with human T and B cells may be used to study whether OKT3 will potentiate anti-tumor immune responses.

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Families Citing this family (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6685941B1 (en) 1988-11-23 2004-02-03 The Regents Of The University Of Michigan Methods of treating autoimmune disease via CTLA-4Ig
DE69032484D1 (de) * 1989-10-27 1998-08-20 Arch Dev Corp Zusammensetzungen und deren verwendung zur förderung der immunopotentiation
US6406696B1 (en) 1989-10-27 2002-06-18 Tolerance Therapeutics, Inc. Methods of stimulating the immune system with anti-CD3 antibodies
US5480895A (en) * 1991-09-27 1996-01-02 New York Society For The Relief Of The Ruptured And Crippled, Maintaining The Hospital For Special Surgery Method of producing antibodies to a restricted population of T lymphocytes, antibodies produced therefrom and methods of use thereof
US20030108548A1 (en) * 1993-06-01 2003-06-12 Bluestone Jeffrey A. Methods and materials for modulation of the immunosuppressive activity and toxicity of monoclonal antibodies
GB9607711D0 (en) * 1996-04-13 1996-06-19 Univ Sheffield T-cell dependent vaccine
WO2000039303A2 (en) 1998-12-31 2000-07-06 Chiron Corporation Modified hiv env polypeptides
AU2487300A (en) 1998-12-31 2000-07-31 Chiron Corporation Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof
US6737398B1 (en) * 1999-09-30 2004-05-18 National Jewish Medical And Research Center Modulation of γδ T cells to regulate airway hyperresponsiveness
EP1246643A4 (de) 1999-10-14 2005-05-11 Jeffery A Ledbetter Dna impfstoffe, welcheein antigen kodieren, dass an eine cd40-bindende domäne bindet
US6797514B2 (en) * 2000-02-24 2004-09-28 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US7572631B2 (en) * 2000-02-24 2009-08-11 Invitrogen Corporation Activation and expansion of T cells
US6867041B2 (en) * 2000-02-24 2005-03-15 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US20030235908A1 (en) * 2000-02-24 2003-12-25 Xcyte Therapies, Inc. Activation and expansion of cells
US20030119185A1 (en) * 2000-02-24 2003-06-26 Xcyte Therapies, Inc. Activation and expansion of cells
US7541184B2 (en) 2000-02-24 2009-06-02 Invitrogen Corporation Activation and expansion of cells
EP1227822A2 (de) * 2000-05-25 2002-08-07 Xcyte Therapies, Inc. Methoden zur wiederherstellung oder verstärkung der immunüberwachung durch t-zellen nach natürlicher oder künstlich induzierter immunsuppression
US8178098B2 (en) 2001-04-03 2012-05-15 National Jewish Health Method to inhibit airway hyperresponsiveness using aerosolized T cell receptor antibodies
AU2002320314A1 (en) 2001-07-05 2003-01-21 Chiron, Corporation Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof
US20030138434A1 (en) * 2001-08-13 2003-07-24 Campbell Robert L. Agents for enhancing the immune response
US20030170614A1 (en) 2001-08-31 2003-09-11 Megede Jan Zur Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
EP2075256A2 (de) 2002-01-14 2009-07-01 William Herman Gezielte Liganden
US20050084967A1 (en) 2002-06-28 2005-04-21 Xcyte Therapies, Inc. Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation
WO2004003142A2 (en) * 2002-06-28 2004-01-08 Xcyte Therapies, Inc. Compositions and methods for restoring immune repertoire in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation
US20040175373A1 (en) * 2002-06-28 2004-09-09 Xcyte Therapies, Inc. Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation
CA2525519A1 (en) * 2003-05-08 2004-12-02 Xcyte Therapies, Inc. Generation and isolation of antigen-specific t cells
EP1663308A1 (de) * 2003-09-22 2006-06-07 Xcyte Therapies, Inc Zusammensetzungen und verfahren zur beschleunigung der hämatologischen erholung
AU2004291107B2 (en) 2003-11-14 2010-09-30 Brigham And Women's Hospital, Inc. Methods of modulating immunity
CA2554978A1 (en) * 2004-02-04 2005-08-25 The Trustees Of Columbia University In The City Of New York Anti-cd3 and antigen-specific immunotherapy to treat autoimmunity
EA010350B1 (ru) 2004-06-03 2008-08-29 Новиммун С.А. Антитела против cd3 и способы их применения
EP2497496A3 (de) 2005-07-11 2013-02-20 Macrogenics, Inc. Verfahren zur Behandlung von Autoimmunerkrankungen mithilfe von immunsuppressiven monoklonalen Antikörpern mit reduzierter Toxizität
EP2354162A1 (de) * 2005-09-12 2011-08-10 Novimmune SA Anti-CD3-Antikorper-formulierungen
EP1937309A4 (de) * 2005-09-14 2010-01-20 Univ Columbia Mit anti-cd3-antikörper induzierte regulierende cd8+-t-zellen
US20070178113A1 (en) * 2005-11-22 2007-08-02 Backstrom B T Superantigen conjugate
NZ573132A (en) * 2006-06-06 2012-05-25 Glaxo Group Ltd Administration of anti-cd3 antibodies in the treatment of autoimmune diseases
BRPI0713426A2 (pt) 2006-06-14 2012-10-09 Macrogenics Inc métodos de tratar, diminuir a progressão, ou melhorar um ou mais sintomas de um distúrbio, e de prevenir ou retardar o inìcio de um distúrbio
KR101865527B1 (ko) 2006-09-01 2018-06-07 가부시키가이샤 니콘 방전램프, 광원장치, 노광장치 및 노광장치의 제조방법
CA2673470A1 (en) * 2006-12-21 2008-07-03 Macrogenics, Inc. Methods for the treatment of lada and other adult-onset autoimmune diabetes using immunosuppressive monoclonal antibodies with reduced toxicity
US20080248025A1 (en) * 2007-03-27 2008-10-09 National Jewish Medical And Research Center Gamma Delta T Cells and Methods of Treatment of Interleukin-17 Related Conditions
NZ580755A (en) † 2007-04-03 2012-05-25 Micromet Ag Cross-species-specific cd3-epsilon binding domain
EP2242513A2 (de) * 2008-01-18 2010-10-27 Hadasit Medical Research Services & Development Ltd. Kombinationstherapie von beta-glykolipiden und antikörpern zur behandlung immunverwandter erkrankungen
US9133436B2 (en) * 2010-02-04 2015-09-15 The Trustees Of The University Of Pennsylvania ICOS critically regulates the expansion and function of inflammatory human Th17 cells
AU2011246893A1 (en) 2010-04-29 2012-11-08 Hadasit Medical Research Service And Development Co. Ltd. Methods and compositions for treating hepatitis with anti-CD3 immune molecule therapy
WO2012012737A2 (en) 2010-07-23 2012-01-26 The University Of Toledo Stable tregs and related materials and methods
US20130273055A1 (en) 2010-11-16 2013-10-17 Eric Borges Agents and methods for treating diseases that correlate with bcma expression
TWI638833B (zh) 2010-11-30 2018-10-21 中外製藥股份有限公司 細胞傷害誘導治療劑
MY202024A (en) * 2012-10-29 2024-03-29 Univ Arkansas Novel mucosal adjuvants and delivery systems
US20150174242A1 (en) 2013-12-19 2015-06-25 Mayo Foundation For Medical Education And Research Monovalent anti-cd3 adjuvants
NZ724710A (en) 2014-04-07 2024-02-23 Chugai Pharmaceutical Co Ltd Immunoactivating antigen-binding molecule
CN106459954A (zh) 2014-05-13 2017-02-22 中外制药株式会社 用于具有免疫抑制功能的细胞的t细胞重定向的抗原结合分子
WO2016059220A1 (en) 2014-10-16 2016-04-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Tcr-activating agents for use in the treatment of t-all
PE20171764A1 (es) 2015-01-23 2017-12-21 Sanofi Sa Anticuerpos anti-cd3, anticuerpos anti-cd123 y anticuerpos biespecificos que se unen especificamente a cd3 y/o cd123
EP3091032A1 (de) 2015-05-08 2016-11-09 Miltenyi Biotec GmbH Cd3-spezifischer, humanisierter antikörper oder fragment davon
WO2017077382A1 (en) 2015-11-06 2017-05-11 Orionis Biosciences Nv Bi-functional chimeric proteins and uses thereof
US11649293B2 (en) * 2015-11-18 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for enhancing humoral immune response
WO2017086367A1 (ja) 2015-11-18 2017-05-26 中外製薬株式会社 免疫抑制機能を有する細胞に対するｔ細胞リダイレクト抗原結合分子を用いた併用療法
CN117024599A (zh) 2016-02-05 2023-11-10 奥里尼斯生物科学私人有限公司 双特异性信号传导剂及其用途
TWI790206B (zh) 2016-07-18 2023-01-21 法商賽諾菲公司 特異性結合至ｃｄ３和ｃｄ１２３的雙特異性抗體樣結合蛋白
EP3576765A4 (de) 2017-02-06 2020-12-02 Orionis Biosciences, Inc. Zielgerichtetes manipuliertes interferon und verwendungen davon
WO2021064069A1 (en) 2019-10-02 2021-04-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment of adult t-cell leukemia/lymphoma
CA3169827A1 (en) 2020-03-10 2021-09-16 Kunwar Shailubhai Compositions of il-6/il-6r antibodies and methods of use thereof
EP4168537A1 (de) 2020-06-17 2023-04-26 Tiziana Life Sciences PLC Zusammensetzungen und verfahren zur verstärkung von t-zelltherapien mit chimärem antigenrezeptor (car)
JP2023535792A (ja) 2020-07-30 2023-08-21 ティジアーナ ライフ サイエンシズ パブリック リミティド カンパニー コロナウイルス治療のための抗ｃｄ３抗体
WO2022221767A1 (en) 2021-04-16 2022-10-20 Tiziana Life Sciences Plc Subcutaneous administration of antibodies for the treatment of disease
WO2023064942A1 (en) 2021-10-14 2023-04-20 Tiziana Life Sciences Plc Methods of suppressing microglial activation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0172107A1 (de) * 1984-08-10 1986-02-19 Praxis Biologics, Inc. Immunogene Konjugate der LT-B Enterotoxinuntereinheit von E. coli und kapselige Polymere
WO1989006974A2 (en) * 1988-02-01 1989-08-10 Praxis Biologics, Inc. T-cell epitope as carriers molecule for conjugate vaccines
EP0336379A2 (de) * 1988-04-04 1989-10-11 Oncogen Limited Partnership Antikörper-Heterokonjugate zur Regulation der Lymphozytenaktivität

Family Cites Families (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS51133489A (en) * 1975-05-14 1976-11-19 Tokyo Daigaku Process for producing microbial components of pseudomonas aeruginosa h aving antimicrobial and antitumor activities
US4658019A (en) * 1979-04-26 1987-04-14 Ortho Pharmaceutical Corporation Complement-fixing monoclonal antibody to human T cells
US4515893A (en) * 1979-04-26 1985-05-07 Ortho Pharmaceutical Corporation Hybrid cell line for producing complement-fixing monoclonal antibody to human T cells
US4361549A (en) * 1979-04-26 1982-11-30 Ortho Pharmaceutical Corporation Complement-fixing monoclonal antibody to human T cells, and methods of preparing same
US4221794A (en) * 1979-06-21 1980-09-09 Newport Pharmaceuticals International, Inc. Method of imparting immunomodulating and antiviral activity
US4395394A (en) * 1979-10-26 1983-07-26 Pfizer Inc. Use of lipid amines formulated with fat or lipid emulsions as vaccine adjuvants
GB8314362D0 (en) * 1983-05-24 1983-06-29 Celltech Ltd Polypeptide and protein products
US4882317A (en) * 1984-05-10 1989-11-21 Merck & Co., Inc. Covalently-modified bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers and methods of preparing such polysaccharides and conjugataes and of confirming covalency
US4695624A (en) * 1984-05-10 1987-09-22 Merck & Co., Inc. Covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers, and methods of preparing such polysaccharides and conjugates and of confirming covalency
US5279960A (en) * 1984-07-05 1994-01-18 Enzon Corp. 25 KD coccidial antigen of eimeria tenella
NZ214503A (en) * 1984-12-20 1990-02-26 Merck & Co Inc Covalently-modified neutral bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins, and methods of preparing such polysaccharides and conjugates
US5078998A (en) * 1985-08-02 1992-01-07 Bevan Michael J Hybrid ligand directed to activation of cytotoxic effector T lymphocytes and target associated antigen
US4676980A (en) * 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US4882424A (en) * 1987-05-11 1989-11-21 Dana-Farber Cancer Institute, Inc. Activation antigen
US5639853A (en) * 1987-09-29 1997-06-17 Praxis Biologics, Inc. Respiratory syncytial virus vaccines
DE68924850T2 (de) * 1988-06-14 1996-10-10 Cell Med Inc Heterofunktionelle zellular immunologische reagenzien, impfstoffe daraus und verwendungsverfahren.
AU4334389A (en) * 1988-10-14 1990-05-01 Trustees Of Columbia University In The City Of New York, The A differentiation antigen, nda5, associated with amplification of differentiation of t and b lymphocytes
IL92382A (en) * 1988-11-23 1994-12-29 Univ Michigan Use of a ligand specific for CD28 in the manufacture of medicament
US5225538A (en) * 1989-02-23 1993-07-06 Genentech, Inc. Lymphocyte homing receptor/immunoglobulin fusion proteins
WO1991001143A1 (en) * 1989-07-14 1991-02-07 Praxis Biologics, Inc. Stable vaccine compositions containing interleukins
SE8903100D0 (sv) * 1989-09-20 1989-09-20 Pharmacia Ab New pharmaceutical agent
DE69032484D1 (de) * 1989-10-27 1998-08-20 Arch Dev Corp Zusammensetzungen und deren verwendung zur förderung der immunopotentiation
GB8928874D0 (en) * 1989-12-21 1990-02-28 Celltech Ltd Humanised antibodies
ZA91463B (en) * 1990-01-25 1992-09-30 Bristol Myers Squibb Co Method of activating cytolytic activity of lymphocytes using anti-cd28 antibody
WO1992000092A1 (en) * 1990-07-02 1992-01-09 Bristol-Myers Squibb Company Ligand for cd28 receptor on b cells and methods
EP0575537A1 (de) * 1991-03-08 1993-12-29 Cytomed, Inc. Lösliche cd28 proteine und behandlungsmethoden damit
US5770197A (en) * 1991-06-27 1998-06-23 Bristol-Myers Squibb Company Methods for regulating the immune response using B7 binding molecules and IL4-binding molecules
ES2123001T5 (es) * 1991-06-27 2009-04-16 Bristol-Myers Squibb Company Receptor ctl4a, proteinas de fusion que lo contienen y sus usos.
AU684461B2 (en) * 1992-04-07 1997-12-18 Regents Of The University Of Michigan, The CD28 pathway immunoregulation
WO1993025712A1 (en) * 1992-06-15 1993-12-23 The Regents Of The University Of California Screening assay for the identification of immunosuppressive drugs
US5394321A (en) * 1992-09-02 1995-02-28 Electric Power Research Institute, Inc. Quasi square-wave back-EMF permanent magnet AC machines with five or more phases
WO1994023760A1 (en) * 1993-04-14 1994-10-27 The United States Of America As Represented By The Secretary Of The Navy Transgenic animal model for autoimmune diseases
US5885573A (en) * 1993-06-01 1999-03-23 Arch Development Corporation Methods and materials for modulation of the immunosuppressive activity and toxicity of monoclonal antibodies
WO1994028912A1 (en) * 1993-06-10 1994-12-22 The Regents Of The University Of Michigan Cd28 pathway immunosuppression
JPH09500788A (ja) * 1993-07-26 1997-01-28 ダナ・ファーバー・キャンサー・インスティテュート・インコーポレイテッド Ｂ７−２：ｃｔｌａ４／ｃｄ２８カウンターレセプター
WO1995005464A1 (en) * 1993-08-16 1995-02-23 Arch Development Corporation B7-2: ctla4/cd28 counter receptor
AUPQ960500A0 (en) * 2000-08-23 2000-09-14 Infinity Beautification Enterprises Pty Ltd A planter box

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0172107A1 (de) * 1984-08-10 1986-02-19 Praxis Biologics, Inc. Immunogene Konjugate der LT-B Enterotoxinuntereinheit von E. coli und kapselige Polymere
WO1989006974A2 (en) * 1988-02-01 1989-08-10 Praxis Biologics, Inc. T-cell epitope as carriers molecule for conjugate vaccines
EP0336379A2 (de) * 1988-04-04 1989-10-11 Oncogen Limited Partnership Antikörper-Heterokonjugate zur Regulation der Lymphozytenaktivität

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 107, no. 15, 12 October 1987, Columbus, Ohio, US; abstract no. 126693z, B.M. SULTZER ET AL.: "IMMUNOMODULATION BY PROTEINS OF BORDETELLA PERTUSSIS." page 32; XP002056038 *
GOV. REP. ANNOUNCE. INDEX (U.S.), vol. 87, no. 5, 1987 *
J. KAPPLER ET AL.: "VBETA-SPECIFIC STIMULATION OF HUMAN T CELLS BY STAPHYLOCOCCAL TOXINS.", SCIENCE, vol. 244, 19 May 1989 (1989-05-19), LANCASTER, PA US, pages 811 - 813, XP002055830 *
J. WHITE ET AL.: "THE VBETA-SPECIFIC SUPERANTIGEN STAPHYLOCOCCAL ENTEROTOXIN B: STIMULATION OF MATURE T CELLS AND CLONAL DELETION IN NEONATAL MICE.", CELL, vol. 56, 13 January 1989 (1989-01-13), CAMBRIDGE, MA, US, pages 27 - 35, XP002055831 *
S. TAMURA ET AL.: "PROTECTION AGAINST INFLUENZA VIRUS INFECTION BY VACCINE INOCULATED INTRANASALLY WITH CHOLERA TOXIN B SUBUNIT.", VACCINE, vol. 6, no. 5, October 1988 (1988-10-01), GUILDFORD GB, pages 409 - 413, XP002056037 *

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JP2546544B2 (ja) 1996-10-23
US6113901A (en) 2000-09-05
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US6143297A (en) 2000-11-07
DE69032484D1 (de) 1998-08-20

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